Process for obtaining extracts from pancreas

ABSTRACT

EXTRACTS OF ANIMAL PANCREAS, USEFUL IN OPOTHERAPHY, RE PREPARED BY REMOVING FOREIGN MATTER, DEBRIS AND FAT FROM THE ASPTICALLY COLLECTED PANCREAS IMMEDIATELY AFTER SLAUGHTERING THE ANIMAL. THE REMAINDER IS THEN VERY RAPIDLY FROZEN TO ABOUT A TEMPERATURE OF -78*C. AND MAINTAINED AT A TEMPERATURE OF ABOUT -20*C. THEREUPON, THE PANCREAS IS CRUSHED AT A TEMPERATURE OF ABOUT 2* C. AND SUBJECTED TO MODERATE DIGESTION AT ROOM TEMPERATURE BY ACITVATED TRYPSIN. UPON REMOVING INSOLUBLE MATTER A CLEAR SOLUTION IS OBTAINED. EUGLOBULINS PRESENT IN THE SOLUTION ARE REMOVED BY ADSORPTION ON AN INSOLUBLE SULFATE WHICH IS THEN REMOVED BY FILTRATION, AND THE RESULTING SOLUTION IS CLARIFIED. FINALLY, THE CLARIFIED SOLUTION IS RAPIDLY FREEZEDRIED AT A TEMPERATURE OF ABOUT -80*C.

PROCESS FOR OBTAINING EXTRACTS FROM PANCREAS Filed May 19, 1972 2Sheets-Sheet l 70 with respect F 1 to normal Serum raZ-e E beforetreatment after treatment i g normal 700 serum /o wit/7 respect tonormal serum rate Z00- m before treatment I 2 I after treatment PROCESSFOR OBTAINING EXTRACTS FROM PANCREAS Filed May 19, 1972 2 Sheets-Sheet 2will? respect to narmal Serum rate E21 e/ore treatment 7 after treatmentrecent cases 0 d of Pancreqtftes Diabetes of myocar-a myocara mfarctfnfar'c' rate United States Patent 3,803,305 PROCESS FOR OBTAININGEXTRACTS FROM PANCREAS Yvonne Thuillier, Paris, France, assignor toLaboratories Albert Rolland, Paris, France Continuation-impart ofapplication Ser. No. 863,696, Oct.

10, 1969, now Patent No. 3,676,551, which is a contiuuation-in-part ofabandoned application Ser. No. 530,325, Feb. 28, 1966, which in turn isa continuationin-part of abandoned application Ser. No. 247,827, Dec.28, 1962. This application May 19, 1972, Ser. No. 229,272 The portion ofthe term of the patent subsequent to July 11, 1989, has been disclaimedInt. Cl. A61k 17/08 US. Cl. 424-110 3 Claims ABSTRACT OF THE DISCLOSUREExtracts of animal pancreas, useful in opotherapy, are prepared byremoving foreign matter, debris and fat from the aseptically collectedpancreas immediately after slaughtering the animal. The remainder isthen very rapidly frozen to about a temperature of 78 C. and maintainedat a temperature of about -20 C. Thereupon, the pancreas is crushed at atemperature of about 2 C. and subjected to moderate digestion at roomtemperature by activated trypsin. Upon removing insoluble matter a clearsolution is obtained. Euglobulins present in the solution are removed byadsorption on an insoluble sulfate which is then removed by filtration,and the resulting solution is clarified. Finally, the clarified solutionis rapidly freezedried at a temperature of about 80 C.

SPECIFICATION This application is a continuation-in-part application ofmy copending application Ser. No. 863,696, filed Oct. 10, 1969, now Pat.No. 3,676,551 which is a continuation-inpart application of myappication Ser. No. 530,325, filed Feb. 28, 1966, and now abandoned,which in turn is a continuation-in-part application of my earlier filedapplication Ser. No. 247,827, filed Dec. 28, 1962, and now abandoned.

This invention relates to pancreas preparations and is particularlydirected towards a new and improved process for preserving the activeprinciples contained in tissue from pancreas, and for making themanallergic for physiological and therapeutic purposes.

BACKGROUND The prior products obtained from the pancreas werecharacterized by notable deficiencies, due to the degrading elfects ofthe methods used in the preparation thereof. These prior methodsresulted in modification or even elimination of essential components ofthe active principles contained in the starting materials, essentiallydue to the following:

( 1) Autolysis of the material;

(2) The use of aqueous-alcoholic, alcoholic, acetonic and similarvehicles, as well as heating in acidic media for precipitating theproteins;

(3) Tyndallization (heating at 70 C. for three days) or autoclaving(heating for one hour at 120 C.) to sterilize the product;

(4) Preservation in an aqueous medium, as a consequence of which theactive principles underwent degradation or modification.

Furthermore, there is no doubt that substances are produced by eachtissue of a normal organism, some of which are known, whilst others arestill unknown, the metabolic ice activity of each substance depending onthe constitution of the tissues, so that it is in fact impossible toidentify precisely in general the active principles of an opotherapicextract. (Thuillier, Active Principles of the Hepatic Gland and TheirFunctional RelationsGeneral Interest of Hepatotherapy, published inPraxis, No. 31, Aug. 1, 1957, pages 676-681.)

It is thus essential that the composition of these complete opotherapicextracts be similar to the composition of the fresh organ, and thatduring their preparation these extracts undergo the least possibledegradation.

Another essential problem in the manufacture of products containingactive principles of animal organs, intended for injection, is thesufiicient elimination of specific heterogeneous proteins, equallycontained in the starting material. When specific proteins of an animalor a determined organ are injected into the human organs, detrimentalphenomena arise, such as anaphylactic shocks and seroreactions, e.g.,urticaria and allergy. The presence of these proteins in productsobtained by the above-described methods thus renders the usefulapplication of the thus preserved active principles impossible.

THE INVENTION The instant invention overcomes the aforesaiddisadvantages of the conventional methods by maintaining the activeprinciples of the pancreas in their natural physiological equillibrium.

The basic operations involved in producing the products of thisinvention are the following:

1) Quick freezing to about -78 C. of the pancreas under sterileconditions immediately after aseptic removal thereof from the freshlyslaughtered animal.

(2) Eifecting a digestion with elimination of the antigenic activity ofthe material so as to render same nonallergenic.

(3) Freeze-drying of the material rendered non-allergenic.

All process steps described hereinbefore are carried out under sterileconditions, thereby avoiding the necessity for subsequent sterilizationof the final pancreatic preparation.

By means of the process according to the invention the integrity of thecellular content of active principles is maintained in its naturalphysiological equilibrium, which is achieved more particularly by thestabilizing action of the cold temperatures which are maintained duringeach step of the preparation.

Moreover, the process according to the invention is also carried outunder the following conditions:

fully protected from a loss up to this stage is thereby preserved.

Furthermore, it is the object of the present process to obtain completeextracts whose content of therapeutically active principles correspondsto the one of the starting material, as regards their nature and theirproportions, which extracts, moreover, do not show any antigenic action,so that they may be injected into humans without danger.

The process of the invention is based on the following experimentalfindings: the antigen contained in the pseudoglobulins and euglobulinsis completely adsorbed by barium or calcium sulfate; thepseudo-globulins and euglobulins are freed from the mass by means of amoderate cleavage of the protein; and the antigenic power is thuseliminated by means of adsorption on barium sulfate.

Theoretically, the advantageous results of the novel process accordingto the invention are in conformity with recent discoveries made duringphysico-chemical studies on enzymes (Heidelberger, Contribution ofImmuno- Chemistry to the Study of Biological Structures, published inBulletin de la Societe de Chimie Biologique, 1964, vol. 46, No. 11, pp.1293-1298), according to which it is possible to maintain the enzymaticactivity of proteic elements when their immunologic activities arediflerent. In proceeding with special caution it is thus possible topreserve the fraction of the active principle of the protein which is ofinterest, and simultaneously to eliminate the antigenic site.

FLOW DIAGRAM OF THE PROCESS ACCORDING TO THE INVENTION Startingmaterial: Pancreas removed aseptically promptly after slaughter;

Quick freezing to 78 C.;

Transport to laboratory in refrigerated containers held at 20" C.;

Mechanical crushing;

Crushings digested at ambient temperature 'by the action of trypsin,activated with calcium ions, and clarified;

pH of clarified liquid reduced to pH 3.7 with H 50 pH released to pH 9by addition of Ba(OH)= or Ca(OH) resulting in precipitation of insolublesulfate with euglobulin aflixed thereto;

Precipitate removed and liquid clarified;

pH of liquid reduced to pH 3.7, and clarified;

Liquid quickly frozen to --80 C.;

Freeze-dried, plate temperature 80" C.; and under a vacuum of 10- mm.Hg;

Final product.

The process of the invention is applicable to the preparation andpreservation of physiologically active materials from pancreatic tissueobtained from any kind of animal. Preferably, mammals such as pigs,sheep and bovine animals or their fetuses in the later stages ofdevelopment are used.

The tissues of pancreas is collected aseptically at the time ofslaughtering, and is freed from foreign portions and various debris(fat) and frozen very quickly to a low temperature, preferably about 78(3., this being done in order to avoid any autolysis or fermentationphenomena. The constituents of the cell are thus preserved withoutdegradation.

The organs are crushed in a mechanical crusher in order to obtain amaximum surface etfect and to simultaneously free the cellular content.

The crushed material is rendered anallergic in two stages:

(a) Moderate cleavage of the proteins by activated trypsin; specificeuglobulins are left in the mass; (b) Fixation of euglobulins on aninsoluble sulfate.

After the disintegration by calcium-activated trypsin, the pH of theexact is first brought to 3.7 with sulfuric acid and then to 9 by theaddition of barium or calcium hydroxide, at a temperature not exceedingroom temperature. The fluid mass is stirred to cause completeprecipitation of the insoluble sulfate which carries down with it theeuglobulins which have been adsorbed thereon.

After complete precipitation of the sulfate with the euglobulinsadsorbed thereon, and removal of the precipitate, the residual solutionis freeze-dried.

The ampoules containing the material are frozen in a drying chamber,cooled to C. (the freezing point of the material being within the rangeof from 10 C. to 15 0.).

During the drying step under reduced pressure the temperature of theplate is kept at 70 C. to 80 C., and the temperature of the coldcondenser is maintained within the range of from C. to C. The pressureof the order of 10" mm. Hg.

The freeze-dried product is obtained as a powder, which can be dissolvedin a pyrogenic distilled water to form an injectable solution.

The above-described process of the present invention is set forth inspecific illustrative embodiments in the following example.

EXAMPLE (a) First operation.Aseptic separation with quick freezing Thestarting material is l-l2 kg. of pancreas, collected from steers lessthan two years old. Freezing to 78 C. is effected immediately afterslaughtering. Each batch of pancreas collected under sterile conditionsis transported to the laboratory in refrigerated containers held at 20C.

(b) Second operation.Mechanical crushing Just before crushing, thepancreases were thawed in their containers by subjecting the containersto a temperature of +2 C. for 12 hours, after which, while still understerile conditions, they were put through the crusher. The crushedorgans were collected.

Cleavage of protein molecules solution was filtered, e.g., over a nyloncloth and then clarified by centrifuging. The solution was 'keptovernight at 5" C.

Elimination of antigenic globulins The solution was acidified by addingsulfuric acid at a dilution ratio of 1/7 by weight, until the pH reached3.9.

Pure barium hydroxide was then added until the pH reached 9. An abundantprecipitate formed.

After standing for at least one hour the suspension was centrifugeduntil a perfectly clear liquid was obtained. If necessary, the liquidmay be filtered over C The pH was then brought to 3.7. The liquid wasagain centrifuged and further clarified by filtration. The absence ofbarium was checked by using sulfuric acid at a dilution ratio of 1/ 7 byweight on a fraction of the liquid. The dry extract should be controlledand should amount to 10% of the liquid. The solution, which should havea pH of from 4.2 to 4.5 was filled into sterile ampoules, eachcontaining 2.5 ml. of said solution.

(c) Third operation The ampoules were frozen by being placed immediatelyin a sterile drying chamber, cooled to 80" C. (Freezing point of theproduct within the range -l0 C. to -15 C.) Rapid freezing took placein afew seconds.

Drying took place in a drying chamber provided with glass windows, whichwas cooled by circulation of alcohol and PRSON-l3. Temperature of theplates was maintained within the range of from 70 C. to -80 C., and thatof the cold condenser within the range of from 90 C. to l00 C. The useof said condenser and of a blade-type rotary pump permitted the vacuumto reach an order of about mm. Mg.

Towards the end, the operation was carried out in an inert atmosphere(nitrogen) to avoid any oxidation.

During the freeze-drying step the product never reached a temperatureexceeding 0 C.

The extract contains tissue hormones, callicrein, vagotonin and otherprinciples present in the pancreas. It is a white powder with total N,10-11%; amino N, 7-8%.

A qualitative analysis of the free and combined aminoacids shows thatthe composition is similar to that of the fresh organ. The absence ofantigenic substances is controlled by the Gucterlony method. The producthas no allergenic eifect. Considerable amounts of desoxyriboncleicacids, organic phosphorus and pentoses are present.

PHARMACODYNAMIC STUDY Numerous anatomo-clinical, biological andexperimental observations which lead to the conclusion that thepancreatic secretions play an important role in the lipoprotein balanceof the serum and in the integrity of the vascular walls (I. Arnal,Atherosclerosis and Pancreatic Secretions, thesis for doctorate inmedicine 1966, Paris), have prompted a study of the influence of thepancreatic extract of the present invention on the evolution of theexperimental arterial lesions.

The process of study chosen was the experimental cholesterolic atheromain the rabbit (P. Gendre; Compt. Rend. Soc. Biol. 162, 412-413 (1968).The atheroma is provoked by subjecting the rabbits, for three months, toa high cholesterol diet composed of a normal diet for rabbits (U.A.R.),plus oil of maize (6%), and cholesterol (2%).

(a) Effect of the pancreas extract on cholesterolemia and on lipemia.

32 rabbits with an average weight of 1.5 kg. were distributed in threegroups:

Group A: 12 rabbits receiving the high cholesterol diet;

Group B: 12 rabbits receiving the high cholesterol diet and in addition,each day by I.M. route, 50 mg. of pancreas extract of the presentinvention per kg. of body weight;

Group C: 8 rabbits receiving only the normal diet (UAR).

Every two weeks, the following determinations were made in the serum:

Total cholesterol (method of Pearson, Stern and Mc- Gavack) Total lipids(method of Chabrol and Charonnat) Esterified fatty acids (method ofStern and Chapiro).

The results obtained were as follows:

In the animals of Group A, receiving the high cholesterol diet, theserum level of total lipids, of total cholesterol and of fatty acidsincrease during the course of the study and reach, after twelve weeks,the values indicated in the following table. In the animals of Group B,the increase is clearly less high, and the differences of the averagesbetween the two groups of rabbits remain significant for all thebiological examinations made after three months of atherogenic diet, asindicated in the following table:

Average of the serum rates after twelve weeks of atherogenic dietControl Treated (Group A) (Group B) Significance Tgtal lipids (g./liter58. 4 44. 3 0. 01 P 0. 02

erum. Total cholesterol (g./liter 27.7 20.8 0.01 P 0.02

serum). Esterified fatty acids 66. 0 55. 4 0. 001 P 0. 01

(meqJliter serum).

The administration to rabbits on an atherogenic diet of the pancreaticextract of the present invention reduces very considerably the increaseof the levels of lipids, cholesterol and esterified fatty acids in theserum.

(b) Effect on the retention of triolein and cholesterol marked with 14C. in the wall of the aorta Group A 6 rabbits receiving *C-triolein;Group A 6 rabbits receiving 4- C-chloesterol; Group B 6 rabbitsreceiving C-triolein; Group B 6 rabbits receiving 4- C-cholesterol;Group C 4 rabbits receiving C-triolein; Group C 4 rabbits receiving 4-C-cholesterol.

All the rabbits, fasting since the injection, were then sacrificed 24hours after the administration of the products marked with C, and threedifferent parts of the aorta were immediately removed (the arch of theaorta, 3 cm. of thoracic aorta and 3 cm. of abdominal aorta).

The fragments of aorta were dissected and washed in physiological saltsolution and then examined histologically under the microscope. Thenthey were dissolved in hydroxide of hyamine for 48 hours and kept at C.in order to measure the activity with the aid of IDL scintillationspectrometer, in liquid medium. After cooling, an accurately weighedaliquot of the hyamine extract was introduced into a counting flask,with 10 ml. of the scintillating Kinard solution.

The different activities expressed in counts per second are given in thefollowing table, in percent of the activity injected, per cm. of aortafor the thoracic and abdominal aorta and per mg. of dried aorta for thearch of the aorta.

Thoracic aorta (3 cm.), Abdominal aorta (3 cm.),

activitylcmpercent Arch of the arota, activity/mg. of dried activity/cm.percent aorta percent injected injected activity injected activityactivity Cholesterol Triolein Cholesterol Trlolein Cholesterol Triolein4-0 14 0 14 4'0 14 4-0 14 4-0 14 0 14 Normal controls (batch C) 0.19-10- 0. 18-10 0. 20-10- 0. 1810- 0. 2940- 0. 23-10 Controls (batch A)after three months of atherogenic diet 2 0. 1840- 0. -10 0. 77-10- 0.46-10 0. 9840- 0. 44-10- Treated (batch B) after three months ofatherogenic diet 2 0. 55-10- 0. 43-10- 0. 46-10- 0. 23-10- 0. 5540- 0.3440- 1 Average of 4 animals for each marked product injected. 2 Average016 animals.

These results show that the transfers of triolein, and especially ofcholesterol into the wall of the aorta are considerably increased withrespect to the normal (batch C) and for the animals having received theatherogenic diet (batch A), for three months, but that this increase inthe transfer of triolein and cholesterol is significantly less in theanimals receiving at the same time the pancreas extract of the presentinvention (batch B).

At the same time, a histological examination shows that theadministration of the pancreas extract of the present invention acts toreduce the thickening of the arterial wall.

BIOLOGICAL AND CLINICAL STUDY This study has shown that the pancreasextract of the present invention is very effective in the treatment ofarterial diseases, pancreatitis and non-insulin dependent diabetes.

It is well known at present that atherosclerosis comes from adisfunctioning of the pancreatic enzymes which control the metabolism ofthe arterial wall.

A number of auhors, including Mr. Perrault (Atherosclerosis andPancreatic Secretion; Coll. Prog. Med. 1967, 95, pp. 335-345) and B.Goodhead (Vascular Factors in Acute Pancreatites"; The Lancet, October18, 1969, pp. 830-831) have emphasized the importance of theatheromatous lesions of the arterial network in the course of chronicpancreatitis, as well as the frequent association of coronary thrombosisand acute pancreatitis. Atherosclerosis is associated with a disturbancein the metabolism of lipids and cholesterol, transported in the bloodand solubilized under the form of slow and rapid lipoproteins. Adisturbance of pancreatic lipases and cholesterol-esterases liberatelipides and cholesterol which deposit and form atheroma plaques. Abiological and clinical study has shown that the pancreas extract of thepresent invention contains the essential enzymatic factors whichregulate lipo-protein disturbances in patients suffering from arterialdiseases, coronary insufliciencies and pancreatitis.

It is known that a disfunction of certain enzymic systems of pancreaticorigin (elastase and collagenase) occurs in the alteration of theelastic fibers and the depolymerization of the mucopolysaccharides ofthe collagen forming the arterial tissue. The biological and clinicalstudy made in a number of cases of arterial diseases has shown that thepancreas extract of the present invention has a role in controlling theelastase-antielastase complex and thus enables the disturbances whichprovoke or favor the arterial hardening and the thickening of arterialwall to be corrected or stabilized.

(a) Role of regulator of the pancreas extract of the present inventionon the lipoproteins of the serum (which are the circulating form oflipids and cholesterol) A study of the serum conveyors, slow and rapidlipoproteins, was made in patients before and after treatment.

By this means, one can obtain a picture of the dynamic interaction ofthe enzymes and antienzymes of pancreatic origin in the metabolism ofthe lipids and correlatively of the carbohydrates.

A study of more than 400 sera by the immunoelectrophoretic method hasdisclosed the existence of two groups of lipoproteins: the more mobilelipoproteins or group a, the lipoproteins of slower mobility or group 8.

By immunoelectrophoretic analysis according to the Scheidegger method(Burtin P., The Proteins of Normal Human Plasma, chapter 5,Immunoelectrophoretic Analysis, edited by P. Grabar and P. Burtin,Amsterdam Elsevier Publishing Company, 1964, p. 120) in veronal agaragarat 1.5%, pH 8.2, and after dyeing by the Soudan W,

the soudanophile precipitation arcs corresponding to the two enumeratedgroups are observed.

The lipidographs, which include slow (,8) and rapid (a) lipoproteinrates and the ratio fi/a have been deter- 8 mined byimmunoelectrophoretic analysis on the serum of patients, before andafter treatment with the pancreas extract of the present invention.

The patients subjected to the treatment presented the followingailments:

(1) Coronary insufficiencies (2) Arterial diseases: atheromatosis,arteriosclerosis (3) Subacute and chronic pancreatitis (hepatopancreatlcmass) (4) Miscellaneous (obesity, considerable astenia, alteration ofthe general state).

The duration of the treatment by injections from 1 to 2 ampoules (eachcontaining 200 mg. of pancreas extract of the present invention dilutedin 5 ml. of solvent at time of use) per day for 20 days was a functionof clinical improvement. RESULTS The results of the lipidographs of somestandard cases have been shown in block tables (Table I: arterialdiseases; Table II arteritis of this lower limb; Table III pancreatitis,diabetes; miscellaneous).

Out of the 30 cases of patients treated with the pancreas extract of thepresent invention, the modifications of the lipidograph were excellentin 3 cases, very good in 13 cases, good in 9 cases, fairly good in 1case and mediocre in 4 cases.

The rate of beta liproproteins was reduced to the benefit of the rate ofalpha lipoproteins; the ratio of the beta/ alpha rate tended to beregulated, i.e., to reconcile the normal serum ratio: 2,4.

On the other hand, the patients thus treated by the pancreas extractwere clinically improved.

(b) Role of regulator of the pancreas extract according to the inventionon the elastase-antielastase complex, in the arterial pathology(hardening of the artery) The pancreas, which is the gland of thelipogenesis and of the lipolysis due to its enzymatic secretions(particularly lipases, proteases (and esterase) is also the gland of theregulation of the elasticity of the artery by means of the elastase thatit secretes.

The elastase has been discovered by two Hungarian authors: S. Balo (Onthe History and Pathogenisis of Atherosclerosis; Geront. Geriat. sectionXX of Excerpta Medica 1959, 2, 279-283) and 1. Range (Isolation andcrystallisation of Elastase From Pancreas of Cattle; Acta Physiol. Acad.Sci. Hung. 1963, 24, 1).

In the course of recent studies, Perrault (Biological Role of theElastase; Pathologie Biol. 1969, vol. 17, pp. 317-326) has shown that,in the serum of pat ie r ts sufiering from atherosclerosis, there is anincreased antielastase activity, whilst the elastase activity is oftendiminished, particularly in chronic cases where pancreatic secretionsare reduced.

In the course of a clinical study on 53 patients suffering fromatherosclerosis, the pancreas extract of the present invention wasadministered to such patients:

By parental route at a rate of 1 or 2 LM. injections per day, of 200 mg.of active ingredient dissolved in 5 ml. of solvent at the moment of use,for 10 to 330 days;

By oral route, for treatments lasting several months, in

the form of drages containing mg. of active ingradient.

The results are given in FIG. 1, 2 and 3 which show the percentages ofincrease (or reduction) of the rates of antielastase and elastase withrespect to the rate, established at 100%, of these enzymes in the normalhuman

